Field of the Invention
The present invention relates to a saccharifying enzyme for biorefinery applications. In particular, the present invention relates to a method for producing cellobiohydrolase with improved activity, and cellobiohydrolase with high activity.
Description of the Related Art
Various modifications of proteins have been performed using transgenic techniques. Also for enzymes, attempts to improve the enzyme activity such as enhancement in activity or increase in expression level have been made. As a method for enhancing the enzyme activity, a method of modifying an amino acid, introducing a mutation into the amino acid at the active center where an enzyme binds to a substrate, and selecting a highly active enzyme has been employed.
In many instances, the substrate binding site of an enzyme is exemplified by a key and a keyhole. Meanwhile, some of enzymes that act on a polymer substrate such as cellulose are known to have a relatively long substrate binding site with a tunnel shape or a groove shape into which the polymer substrate is fitted, though they are limited to only a part of the enzymes.
Exoglucanase that is an enzyme configured to cut the cellulose chain at the end produces cellobiose as a reaction product, and is called cellobiohydrolase. In the case of cellobiohydrolase, the active center has a tunnel shape, as described above, and the cellobiose cut by the enzyme reaction leaves from the opposite side of the tunnel. Therefore, the enzyme reaction proceeds without the cellobiohydrolase separating from the cellulose chain.
Attempts to improve the enzyme activity of cellobiohydrolase have been made in the past, and several mutants have been already reported. For example, a mutant relating to Talaromyces (see the specification of U.S. Pat. No. 8,790,894) and a mutant of cellobiohydrolase derived from Phanerochaete (see Japanese Patent Laid-Open No. 2010-046034) are known.